B2A, a receptor modulator, increases the growth of pluripotent and preosteoblast cells through bone morphogenetic protein receptors.

B2A (B2A2-K-NS) is a synthetic, multidomain peptide which is being developed to augment spinal fusion and bone repair locally. Using pluripotent mesenchymal cells of murine and human origin, we show that B2A-induced cell proliferation in a modest but dose-dependent manner. However, essentially all human tumor lines tested were not responsive or were weakly responsive to B2A. B2A treatment activated extracellular signal-regulated kinases 1 and 2 (ERK1/2), and the proliferation was partially blocked by an mitogen-activated protein kinase (MEK) inhibitor. The bone morphogenetic protein (BMP) type I receptor kinase inhibitors depressed B2A-induced proliferation. Upregulation of bone morphogenetic protein 2 was not involved, as noggin, DAN, or chordin did not block B2A-induced proliferation. These data suggest that B2A-induced proliferation results from cell-type-specific activation of bone morphogenetic protein receptor, which, in turn, regulates ERK1/2 activity. B2A-induced proliferation, acting through ERK1/2, is a phenomenon that, while not strictly related to the ability of B2A to augment BMP-induced differentiation via the small mothers against decapentaplegic pathway, may ultimately contribute to bone repair in vivo.

Skeletal effects of fibroblast growth factor mimetic (F2A) in ovariectomized rats.

The current study was designed to determine whether short-term, systemic treatment with F2A, a mimetic for FGF-2, has skeletal effects in ovariectomized (OVX) rats and adverse side effects in non-skeletal tissues. Groups of sham-operated and OVX rats were maintained untreated for 6 weeks postOVX. These groups (N=6) were then treated IP with vehicle or F2A (0.3, 1.0, or 3.0 mg/kg) daily for 14 days. Histomorphometric analyses were performed in the proximal tibial metaphyses. Hematocrit was normal in F2A-treated OVX rats. Although organ function was not evaluated, histological examination of several organs did not detect any abnormalities. F2A treatment did not increase cancellous bone mass regardless of dose, but OVX rats treated with 1 mg/kg did exhibit increased osteoclast surface. All 3 doses of F2A induced a modest increase in cancellous bone formation. Therefore, F2A appears to increase both cancellous bone resorption and formation, but these skeletal processes are in balance so that, unlike FGF-2, cancellous bone mass is not augmented. However, F2A did not induce the anemia and impaired bone mineralization associated with FGF-2. Therefore, local application of F2A for orthopedic procedures would presumably have minimal side effects, even if the peptide is released to the systemic circulation.

Augmentation of osseous phenotypes in vivo with a synthetic peptide.

The synthetic peptide B2A2-K-NS augmented the in vitro expression of osseous phenotypes when cells were stimulated with BMP-2, an osteoinductive growth factor. B2A2-K-NS significantly enhanced the effects of BMP-2-induced alkaline phosphatase activity and mineralization. In the absence of BMP-2, B2A2-K-NS did not have an effect on these endpoints. Based on these observations, in vivo studies were conducted to evaluate if B2A2-K-NS could augment osseous phenotypes in an osteoinductive environment in which BMP-2 should be present. In one study, human demineralized bone matrix (DBM) was used to generate an osteoinductive environment and the effects of B2A2-K-NS on ectopic mineralization of subcutaneous implants evaluated. In the second study, a noncritical sized defect in rabbit ulnas with inherent reparative capacity was used as the osteoinductive environment and was treated with or without B2A2-K-NS. In the DBM studies, B2A2-K-NS augmented mineralization as determined using a combination of radiographic analysis and von Kossa staining at 4 weeks postimplant. In the rabbit ulna model, B2A2-K-NS significantly increased the radiographic bone density in the defects compared to carrier-only or no-treatment controls after 6 weeks. Histological staining confirmed that B2A2-K-NS generated a pronounced bone repair response. The results are consistent with the hypothesis that B2A2-K-NS augments osseous phenotypes in an osteoinductive environment, and suggests that B2A2-K-NS may have clinical utility.

Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide.

Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.

Synthetic peptide F2A4-K-NS mimics fibroblast growth factor-2 in vitro and is angiogenic in vivo.

A multi-domain synthetic peptide, F2A4-K-NS, mimicked the action of recombinant human FGF-2 (rhFGF-2) in vitro and in an in vivo model of angiogenesis. Like rhFGF-2, F2A4-K-NS was quantitatively shown to bind to FGF receptors in a cell-free receptor binding assay using a chimeric FGFR1 (IIIc)/Fc as monitored by surface plasmon resonance (SPR), and also shown to bind to heparin using biotinylated low-molecular weight heparin in a similar SPR assay. In vitro, F2A4-K-NS triggered signal transduction as monitored by the stimulation of ERK1/2 phosphorylation in human umbilical cord endothelial cells. In cell based assays, it increased cell migration, cell proliferation, and gelatinase secretion; endpoints associated with FGF-2 stimulation. Furthermore, these in vitro effects were mediated with quantities of F2A4-K-NS that were similar to those of rhFGF-2. In vivo, F2A4-K-NS was angiogenic at doses of 40 and 400 ng/implant in a subcutaneous implant assay as determined by morphologic scoring, hemoglobin content, and histology. These results support the hypothesis that F2A4-K-NS is a mimetic of FGF-2 that can substitute for FGF-2 in vitro and in vivo. A synthetic mimetic of FGF-2, such as F2A4-K-NS, could be a useful tool in studying mechanisms of cell activation and potentially in various therapeutic applications.

Radiolabeling brachytherapy sources with Re-188 through chelating microfilms: stents.

Rhenium-188 (Re-188, T(1/2) = 17 h) emits beta particles (E(max) = 2. 12 MeV) having an ideal range for intravascular brachytherapy and certain cancer brachytherapies. Re-188 was attached to metal wafers and stents via a chelating microfilm, and these brachytherapy sources characterized in vitro and in vivo. To prepare the sources, a siloxane film containing reactive amines was plasma deposited on the metal, a chelating microfilm conjugated to the amines, and the chelating microfilm used to attach Re-188. Re-188 was selectively bound to materials coated with the chelating microfilm. Binding correlated with the amount of radionuclide used. Wafers (1 cm(2)) bound up to 62.9 MBq (1.7 mCi) of Re-188 with yields generally near 30%. Stents bound up to 26.6 MBq (720 microCi). Typically, stents were labeled to bind 4-12 MBq and deposit 10-30 Gy at 2 mm in the arterial wall. In phantom studies, the longer nitinol stents deposited doses of 2.3 Gy/MBq (0.085 Gy/microCi), while shorter stainless steel stents deposited 4.62 Gy/MBq (0.171 Gy/microCi). After placement in arteries of pigs, only the Re-188-stents were detected by scintigraphy at times up to 24 h. Scintigraphy did not detect activity in other organs. Blood sampling (0.1-24 h) detected maximum radioactivity (up to 388 cpm/mL/100micro Ci) at 6 h. We conclude that on-demand radiolabeling of stents and other brachytherapy sources with Re-188 can be performed routinely.

188Re- and 99mTc-MAG3 as prosthetic groups for labeling amines and peptides: approaches with pre- and postconjugate labeling.

Either radiolabeled Tc-99m- or Re-188-labeled MAG3-4-nitrophenylester or unlabeled Bz-MAG3-4-nitrophenylester was reacted with amines and peptides to follow a pre- or a postconjugate radiolabeling route, respectively. The model compounds were N'-t-butyloxycarbonyl-1,6-diaminohexane (DH-Boc) and a Lys-protected derivative of the somatostatin analog RC-160 (cyclic D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2). In the case of labeling DH-Boc, both the preconjugate labeling and the postconjugate labeling were found by using analytical HPLC to provide identical radiolabeled compounds regardless whether Re-188 or Tc-99m was used. The results are supported by infrared and mass-spectral data obtained from compounds synthesized using stable rhenium. The 188Re- or 99mTc-MAG3-RC-160 somatostatin analog were synthesized following the preconjugate labeling route and subsequent removal of the protecting group. Biodistributions of 188Re-and 99mTc-MAG3-RC-160 were evaluated in normal and tumor-bearing mice, and were similar to those of radioiodinated 131-RC-160. All radiolabeled analogs of RC-160 were rapidly cleared from the blood and were excreted through the hepatobiliary system with very little normal organ uptake. The tumor uptake (PC-3, human prostate adenocarcinoma) of systemically administered Re-188-MAG3-RC160 was very low, and it reached only 0.28% injected dose/g (%IDg) at 24 h postinjection, similar to what was obtained with I-131-RC-160. Intratumor injections resulted in significant tumor retentions (9.3% ID/g at 24 h).

Increased binding to sigma sites of N-[1'(2-piperidinyl)ethyl)-4-[I-125]-iodobenzamide (I-125-PAB) with onset of tumor cell proliferation.

This study evaluated if the density of sigma sites was modulated following stimulation of mitosis and progression through the cell cycle. The sigma ligand N-[1'(2-piperidinyl)ethyl)-4-[I-125]-iodobenzamide (I-125-PAB) was a binding probe on the mammary tumor cell lines T47D and MCF-7, and the prostate tumor cell line DU-145. Cells at low density and in log phase growth bound more I-125-IPAB than those at high density at or the near plateau phase. Stimulation of mitosis with insulin or fresh 10% serum increased I-125-IPAB binding in all three cell lines. In cell-cycle synchronized cells, the highest amount of binding was found in cells treated with colcemid to block cells in the M-phase, while the lowest amount of binding was found in cells treated with low serum to block the cells in G1. Cells treated with aphidicolin to block cells at G1/S also bound less than cells block in the M-phase. Collectively, these results support a direct correlation between I-125-PAB binding and proliferative status, and suggest an up-regulation of sigma binding sites prior to mitosis.

Localization of colorectal carcinoma by rhenium-188-labeled B72.3 antibody in xenografted mice.

In order to evaluate the feasibility of 188Re-labeled antibodies for radioimmunotargeting, monoclonal antibody B72.3, recognizing TAG-72, expressed on the surface membranes of colorectal cancer cells, was directly labeled with 188Re, obtained from a 188W/188Re generator, using stannous tartrate and compared with 125I-labeled B72.3. As a control, a human IgG was also radiolabeled with 188Re and 125I. Prepared antibodies for 188Re labeling could be stored as kits. Biodistribution was determined in nude mice inoculated with human colorectal carcinoma LoVo. Labeling efficiency and immunoreactivity of 188Re-B72.3 were 80.3% and 64.7%, respectively. 188Re-B72.3 localized specifically in the LoVo tumors. Although the absolute tumor accumulation level of 188Re-B72.3 was lower than 125I-B72.3, 188Re-B72.3 demonstrated higher tumor-to-blood contrast than the 125I-labeled counterpart, 2.04 +/- 0.44 vs. 1.05 +/- 0.28 at 96 hours, because of fast clearance from the blood. 188Re-B72.3 seemed efficient for the imaging and therapy of colorectal carcinoma.

Clinical aspects of local and regional tumor therapy with 188Re-RC-160.

Somatostatin-receptors have been found to be overexpressed in a variety of neuro-endocrine and epithelial cancers. While the introduction of a long-acting somatostatin-analogue, octreotide, exerted mainly anti-cancer activity in neuro-endocrine tumors, no convincing results have been demonstrated in other cancers. RC-160, another somatostatin-analogue has been selected because of its high receptor affinity and its anti-cancer activity. 188Re is a generator produced radionuclide with favourable gamma and beta-emission, allowing diagnostic and therapeutic application. The results of in vivo biodistribution and therapeutic outcome following systemic, intralesional and intracavitary application in animal studies employing 188Re-RC-160 are summarized. Safety considerations, dosimetry estimates and applicable indications are outlined. The clinical impacts of this radiopharmaceutical in cancer management are discussed.

Availability of rhenium-188 from the alumina-based tungsten-188/rhenium-188 generator for preparation of rhenium-188-labeled radiopharmaceuticals for cancer treatment.

Rhenium-188 (beta- = 2.2 MeV; gamma = 155 keV; T1/2 16.9 hours) is an attractive therapeutic radioisotope which is produced from decay of the reactor-produced tungsten-188 parent (T1/2 69 days) and thus conveniently obtained on demand by elution from the alumina-based tungsten-188 /rhenium-188 generator system. The rhenium-188 is obtained as sodium perrhenate by elution of the generator with 0.9% saline. The post elution use of disposable tandem, ion-exchange columns is a simple method for the concentration of rhenium-188 saline solutions with specific volumes > 500 mCi/ml. This method can also extend the useful shelf-life of the generator, which can be as long as one year. The long useful shelf-life of the generator is expected to provide rhenium-188 at very reasonable costs for routine preparation of a variety of radiopharmaceuticals for the treatment of a variety of cancers including breast cancer. We are evaluating two types of Re-188-labeled agents under investigation which have potential for the treatment of breast cancer. Rhenium-188-labeled hydroxyethylidenediphosphonate (HEDP) and Re-188-dimercaptosuccinic acid (DMSA) are being applied for palliative treatment of pain associated with skeletal metastases, and the Re-188-RC-160 somatostatin analogue [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Lys-Val-Cys-Trp-NH2] for somatostatin-receptor-positive tumors. The results of initial clinical studies with the two bone pain agents demonstrate good targeting to skeletal metastases, and use of Re-188-HEDP has resulted in pain palliation with minimal bone marrow suppression in the initial patient studies. While these initial studies have been conducted in patients with prostate cancer, similar results are expected in planned studies in breast cancer patients. In animal studies, Re-188-RC-160 has been successfully used for the local/regional treatment of experimental breast cancer and other cancers. Re-188-RC-160 binds to somatostatin-receptor-positive cells both in vitro and in vivo, including breast cancer cells (ZR-75-1 breast carcinoma and NCI-H69 human small cell ling carcinoma), but not to binding-negative cells (Raji, Burkitt's lymphoma). A structurally similar Re-188-cyclic peptide with different binding specificity (CTOP [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Orn-Thr-Pen-Thr-ol]; an opiate-receptor antagonist) did not bind to target cells. Both gentisic acid and ascorbic acid are present in the Re-188-HEDP and Re-188-RC-160 formulations, and have been found to also significantly reduce radiolytic degradation of the somatostatin peptide analogues, and may have general application in the stabilization of Re-188-labeled radio-pharmaceuticals.

Pre-clinical experience with Re-188-RC-160, a radiolabeled somatostatin analog for use in peptide-targeted radiotherapy.

The clinical potential of radiolabeled peptides such as octreotide and VIP has been widely established for tumor localization. Radiotherapy based on the tumor binding potential of the peptides and the radiotoxic effects of beta- or a-emitting radionuclides is an extension of such applications. Rhenium-188 (T1/2 16.9 hr, beta-max 2.1 MeV) coupled to the analogue RC-160 has been used to establish the feasibility of treating tumors with radiolabeled peptides, and our experience with this approach is summarized. In three different experimental tumor models (human prostate, mammary gland, and small cell lung carcinomas) in nude mice, treatment resulted in significant reduction or elimination of tumor burden. Two routes of administration were used: intra-lesional injection (prostate carcinoma) and intra-cavity injection (mammary and SCLC). Re-188-labeled negative control peptides bound to tumor cells to a low extent and did not exhibit therapeutic benefit. RC-160 by itself did not result in therapeutic benefit. Tumors which did not bind Re-188-RC-160 did not evidence a therapeutic benefit. Uncoupled Re-188 (control) was rapidly excreted via the urinary bladder and did not accumulate in either tumors or normal tissues even following direct injection. Instant radiolabeling kits containing 200 micrograms of RC-160 were labeled with < 3000 MBq of Re-188 in 30 minutes with no need for subsequent purification. These studies establish the conceptual feasibility of targeted radiotherapy based on the local or regional administration of radiolabeled peptides.

Effects of induction of multi-drug resistance on accumulation of 99mTc-sestamibi in vitro.

The aim of our study was to evaluate the speed of induction of drug-resistance and its effects on intracellular MIBI accumulation in established human cell lines in-vitro. Four out of 5 cell lines were sensitive to vincristine, 1 cell line was vincristine-resistant. All vincristine sensitive cell lines became vincristine-resistant following drug exposure. Resistance in low-drug concentrations occurred as early as 24 hours after exposure and progressed to a roughly 1000-times enhanced resistance within 7 days. Induction of drug-resistance was associated with significantly decreased MIBI accumulation in 2 cell lines but was uneffected in 1 cell line, that was primarily drug sensitive as well as in one cell line that was primarily drug-resistant. Our data indicate, that induction of MDR is an extremely rapid process and the development of drug resistance is not always associated with enhanced MIBI extrusion.

Preparation of 188Re-RC-160 somatostatin analog: a peptide for local/regional radiotherapy.

Conditions are described for the preparation of 188Re-RC-160, a radiolabeled synthetic peptide derived from an analogue of somatostatin, using a lyophilized labeling kit containing RC-160 (200 micrograms) and stannous tartrate to which is added carrier-free 188Re (beta, Emax, 2.12 MeV; gamma, 155 keV, 10%; T1/2 = 16.7 h). The kits have been radiolabeled with > 2960 MBq (80 mCi) with a high labeling efficiency and no need for subsequent purification. Radiolabeling results in one major peak when analyzed by reverse-phase (RP) HPLC. Presumptive radiolysis was detected at 2.5 h post-labeling; however, a convenient method is described for stabilizing the kits against radiolysis by the post-labeling addition of ascorbic acid.

Targeting peptides for pleural cavity tumor radiotherapy: specificity and dosimetry of Re-188-RC-160.

Re-188-RC-160, a radiolabeled somatostatin analogue, is being explored for its potential as a local/regionally administered radiotherapeutic agent targeting somatostatin receptor-positive tumors. In studies in vitro and in vivo, Re-188-RC-160 was found to bind to somatostatin receptor-positive cells (NCI-H69, human small cell lung carcinoma), but not to binding-negative cells (Raji, Burkitt's lymphoma). The comparative binding of Re-188-labeled RC-160 [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Lys-Val-Cys-Trp-NH2] or CTOP [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Orn-Thr-Pen-Thr-ol], a mu-opioid receptor antagonist used a negative control compound, was also determined in vitro and in vivo using NCI-H69 cells as targets. Re-188-RC-160 demonstrated higher net binding in vitro and in vivo compared to Re-188-RC-CTOP, and in vitro its binding could be reduced by excess unlabeled RC-160 whereas binding of Re-188-CTOP could not be reduced. Using another somatostatin receptor-positive human tumor line, ZR-75-1, a substantial amount of the cell-bound Re-188-RC-160 was found to be internalized. Does estimates for Re-188-RC-160 in animals containing NCI-H69 tumors indicated that with three serial injections therapeutic doses greater than 60 Gy can be achieved.

Prognostic value of thyroglobulin after thyroidectomy before ablative radioiodine therapy in thyroid cancer.

UNLABELLED: Serum thyroglobulin (Tg) is a suitable marker for differentiated thyroid carcinoma after total thyroid ablation by surgery and 131I therapy. Before the first 131I treatment, Tg is not a reliable tumor marker since it can also originate from remnant tissue. It was hypothesized that the ratio of serum Tg to 131I uptake in the thyroid bed could be used to correct Tg values for variations in remnant tissue. METHODS: The hypothesis was evaluated in 111 patients with differentiated thyroid cancer (38 follicular/73 papillary). Tg and 131I uptake in the thyroid bed were measured before the first 131I therapy. The ratio of Tg to 131I uptake was determined in four groups: Group A, tumor free (n = 81); Group B, lymph node metastases (n = 11); Group C, distant metastases (n = 11); Group D, later recurrence [during a mean follow-up of 56 mo; (n = 8)]. Wilcoxon two-sample test was performed to determine statistical significance between Group A and Groups B-D. RESULTS: Significant differences in the Tg/131I uptake ratios (median) between Group A (1.0 ng/ml/%) and Groups B (3.3 ng/ml/%) and D (3.3 ng/ml/%) were observed (p < 0.01). In tumor-free patients (Group A), there was no value higher than 5.7 ng/ml/%. Therefore, a higher ratio, observed in 14 of the 30 remaining patients, was indicative of metastases or later recurrence. CONCLUSION: The ratio of serum Tg to 131I uptake in the thyroid bed might be used as a prognostic marker for thyroid cancer before implementing ablation with 131I.

Localization of small-cell lung cancer xenografts with iodine-125-, indium-111-, and rhenium-188-somatostatin analogs.

We examined the potential of radiolabeled somatostatin analogs, 125I-Tyr-3-octreotide (125I-octreotide), (111)In-DTPA(diethylenetriaminepentaacetatic acid)-D-Phe-1-octreotide (111In-octreotide), and 188Re-octreotide for targeting small-cell lung cancer (SCLC) in a mouse model. Tyr-3-octreotide was labeled with 125I by the chloramine T method, and (111)In-octreotide was obtained as a kit, while 188Re was eluted from a 188W/188Re generator, and octreotide was directly labeled with 188Re by reducing disulfide bonds. The 125I-, 111In-, and 188Re-octreotides were injected i.v. into athymic mice bearing NCI-H69 tumors, and the biodistributions were determined at 15 min, and 2, 4, 8, and 24 h. Tumor uptakes were 0.5+/-0.2, 0.3+/-0.1, 0.3+/-0.1 %ID/g, and tumor-to-blood ratios were 1.8, 11.9, 1.2 at 8 h for 125I-, 111In-, and 188Re-octreotides, respectively. Accumulations of 111In-octreotide in normal tissues were lower than those of 125I- and 188Re-octreotides. 188Re-octreotide can be used to localize SCLC lesions as efficiently as radioiodinated octreotide. However, 111In-octreotide was the most suitable agent to obtain high tumor-to-normal tissue contrast for localizing SCLC.

Experimental radiotherapy of receptor-positive human prostate adenocarcinoma with 188Re-RC-160, a directly-radiolabeled somatostatin analogue.

The therapeutic potential of the somatostatin analogue RC-160 radiolabeled with 188Re was evaluated in nude mice bearing xenografts of human prostate adenocarcinoma. 188Re-RC-160 was selectively retained in both DU-145 and PC-3 tumors following direct intra-tumor injection at all time points examined (2, 6 and 24 hr post-injection). Unbound 188Re-RC-160 was rapidly excreted via the hepatobiliary system and, with the exception of the gastrointestinal tract, very little normal organ uptake was found at any time point examined. Negative control compounds, 188Re-perrhenate and 188Re-mercaptoacetyl-triglycine (188Re-MAG3), were essentially washed out of the tumor by 6 hr post-injection and were rapidly excreted through the kidneys. 131I-RC-160, used as reference compound, had a biodistribution in tumor-bearing animals similar to that of 188Re-RC-160. In PC-3 xenografts, 188Re-RC-160 gave a dose-dependent therapeutic response (stasis or regression) even in animals with relatively large tumor masses (greater than 600 mm3), whereas the macro-aggregated form of 188Re-RC-160 did not. Long-term studies with 188Re-RC-160 demonstrated a protracted reduction of tumor volume and a positive effect on animal survival. Neither RC-160 by itself nor a 188Re-labeled peptide, unrelated to somatostatin (PA-22-2, a laminin peptide), demonstrated the reduction in tumor mass observed with 188Re-RC-160. 188Re-RC-160 shows potential as a new clinical agent for treatment of somatostatin-receptor-positive cancers.

Radiotracer binding to brain microsomes determined by thin-layer chromatography.

A thin-layer chromatography (TLC) assay was developed to monitor the interaction of radiotracers with brain microsomes. Murine brain microsomes were coated onto a zone of a TLC strip, the unreacted sites blocked with gelatin, and the radiotracers chromatographed over the microsomes. Radiotracers bound to the microsomes and were separated from the unreacted materials which migrated at or near the solvent front. Up to 80% of the applied radioactivity bound to the brain microsomes when using 99mTc-(d,l) hexamethyl-propyleneamine oxime (HMPAO) and 123I-(S)-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]-benzamide (123I-IBZM) as tracers. On the other hand, the presumptive negative control materials p-I-15-phenyl-pentadecanoic acid-123I (123I-IPPA) and 99mTc-mercapto-acetyl triglycine (MAG3) bound poorly (7% and 4%, respectively). 99mTc-ethyl cysteinate dimer (ECD) interacted poorly (9.9%), a result thought to be consistent with its known inability to be metabolized by nonprimate brain tissue. Radiolabeled octreotide analogues (radiolabeled with 111In, I-131 or 99mTc) also bound, and the binding could be reduced by excess unlabeled octreotide. Also, chemical modification by acylation of Lys5 in 111In-labeled octreotide led to decreased binding (approximately 70%) compared to the original radiotracer. Chromatography of the various radiotracers over TLC strips coated only with gelatin was used to monitor nonspecific binding and was low and frequently below 5%. This technique does not require wash steps or centrifugation, and assays are rapidly completed. The assay could be useful in monitoring the interaction of radiotracers with brain microsomes and in evaluating and developing new radiotracers.

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography.

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.